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This work investigated the healing properties of proteins extracted of latex (HdLP) on excisional wounds. Cell toxicity of HdLP was investigated carried out in murine fibroblasts after incubation with HdLP (12.5-100 µg/ml). The dermal irritability test was performed to evaluate dermal reactions. The wounds were performed and treated with vehicle or HdLP (0.5 %, 1.0 %, and 2.0 %). The macroscopic parameters, histological analysis and measurement of inflammatory markers and mediators were evaluated. HdLP did not exhibit cytotoxicity and did not induce skin irritation. HdLP stimulated the release of IL-1ß at the beginning of the inflammatory phase. This effect probably favored the earlier release of IL-10 by macrophages, during the proliferative phase. The shortening and completeness of healing were characterized by fibroblast proliferation and the presence of newly synthesized collagen fibers. This was accompanied by well-organized re-epithelialization. The involvement of latex proteins in this activity is reported for the first time.
RESUMO
Extracellular vesicles (EVs), released from all cells, are essential to cellular communication and contain biomolecular cargo that can affect recipient cell function. Studies on the effects of contractile activity (exercise) on EVs usually rely on plasma/serum-based assessments, which contain EVs from many different cells. To specifically characterize skeletal muscle−derived vesicles and the effect of acute contractile activity, we used an in vitro model where C2C12 mouse myoblasts were differentiated to form myotubes. EVs were isolated from conditioned media from muscle cells at pre-differentiation (myoblasts) and post-differentiation (myotubes) and also from acutely stimulated myotubes (1 h @ 14 V, C-Pace EM, IonOptix, Westwood, MA, USA) using total exosome isolation reagent (TEI, ThermoFisher (Waltham, MA, USA), referred to as extracellular particles [EPs]) and differential ultracentrifugation (dUC; EVs). Myotube-EPs (~98 nm) were 41% smaller than myoblast-EPs (~167 nm, p < 0.001, n = 8−10). Two-way ANOVA showed a significant main effect for the size distribution of myotube vs. myoblast-EPs (p < 0.01, n = 10−13). In comparison, myoblast-EPs displayed a bimodal size distribution profile with peaks at <200 nm and 400−600, whereas myotube-Eps were largely 50−300 nm in size. Total protein yield from myotube-EPs was nearly 15-fold higher than from the myoblast-EPs, (p < 0.001 n = 6−9). Similar biophysical characteristics were observed when EVs were isolated using dUC: myotube-EVs (~195 nm) remained 41% smaller in average size than myoblast-EVs (~330 nm, p = 0.07, n = 4−6) and had comparable size distribution profiles to EPs isolated via TEI. Myotube-EVs also had 4.7-fold higher protein yield vs. myoblast EVs (p < 0.05, n = 4−6). Myotube-EPs exhibited significantly decreased expression of exosomal marker proteins TSG101, CD63, ALIX and CD81 compared with myoblast-EPs (p < 0.05, n = 7−12). Conversely, microvesicle marker ARF6 and lipoprotein marker APO-A1 were only found in the myotube-EPs (p < 0.05, n = 4−12). There was no effect of acute stimulation on myotube-EP biophysical characteristics (n = 7) or on the expression of TSG101, ARF6 or CD81 (n = 5−6). Myoblasts treated with control or acute stimulation−derived EPs (13 µg/well) for 48 h and 72 h showed no changes in mitochondrial mass (MitoTracker Red, ThermoFisher, Waltham, MA, USA), cell viability or cell count (n = 3−4). Myoblasts treated with EP-depleted media (72 h) exhibited ~90% lower cell counts (p < 0.01, n = 3). Our data show that EVs differed in size, distribution, protein yield and expression of subtype markers pre vs. post skeletal muscle−differentiation into myotubes. There was no effect of acute stimulation on biophysical profile or protein markers in EPs. Acute stimulation−derived EPs did not alter mitochondrial mass or cell count/viability. Further investigation into the effects of chronic contractile activity on the biophysical characteristics and cargo of skeletal muscle−specific EVs are warranted.
RESUMO
BACKGROUND: Exercise is associated with health benefits, including the prevention and management of obesity. However, heterogeneity in the adaptive response to exercise training exists. Our objective was to evaluate if changes in extracellular vesicles (EVs) after acute aerobic exercise were associated with the responder phenotype following 6-weeks of resistance training (RT). METHODS: This is a secondary analysis of plasma samples from the EXIT trial (clinical trial#02204670). Eleven sedentary youth with obesity (15.7 ± 0.5 yrs, BMI ≥95th percentile) underwent acute exercise (60% HRR, 45 min). Blood was collected at baseline [AT0 min], during [AT15-45 min], and 75 min post-recovery [AT120], and EVs purified using size exclusion chromatography from extracted plasma. Afterward, youth participated in 6-weeks RT and were categorized into responders or non-responders based on changes in insulin sensitivity. RESULTS: We assessed EV biophysical profile (size, zeta potential, protein yield, and EV subtype protein expression) in a single-blind fashion. Overall, there was a general increase in EV production in both groups. Average EV size was larger in responders (~147 nm) vs. non-responders (~124 nm; p < 0.05). EV size was positively associated with absolute change in Matsuda index (insulin sensitivity) following RT (r = 0.44, p = 0.08). EV size distribution revealed responders predominantly expressed EVs sized 150-300 nm, whereas non-responders expressed EVs sized 50-150 nm (p < 0.05). At baseline, responders had ~25% lower TSG101, ~85% higher MMP2 levels. EV protein yield was higher in responders than non-responders at AT15 (p < 0.05). CONCLUSIONS: Our data suggest that youth with obesity that respond to RT produce larger EVs that are TSG101+ and CD63+, with increased EV protein yield during acute exercise.